Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

• the present invention relates to a method suitable for routine diagnostics for determining the release of the peptide neurotensin into the circulation.
• Neurotensin is a tridecapeptide found in various tissues and in the circulation of various mammals, including humans, whose primary structure appears to be identical in all mammals. It has the amino acid sequence listed as SEQ ID NO: 2 in the Sequence Listing.
• Neurotensin like most biologically active peptides (5, literature references in the form of numbers in parentheses refer to the attached bibliography), is formed by enzymatic processing from a precursor molecule known as preproneurotensin (with signal sequence) resp. Proneurotensin (PNT, without signal sequence) is called.
• the preproneurotensin / proneurotensin of the mammal contains, in addition to the neurotensin (NT), another biologically active peptide, the hexapetptide Neuromedin N (NMN, see SEQ ID NO: 3).
• Neurotensin precursor peptides have been reported. a. isolated from canine, bovine and rodent tissues and finally also from human tissues.
• the precursor molecules ie the prepro or proneurotensins
• the precursor molecules ie the prepro or proneurotensins
• the precursor molecules ie the prepro or proneurotensins
• the preproneurotensin in the rat or bovine z. B. only 169 amino acids
• the human preproneurotensin has 170 amino acids (17).
• the nucleotide and amino acid sequences of the human pre-proneurotensin have been known for some years (SEQ ID NO: 1, see also US Pat. No. 6,274,720 Bl), as well as those of dog (9), rat (17). and Rind (17, see also the comparison of different Preproneurotensinsequenzen in Figure 2 of the above-mentioned US Patent).
• the amino acids 1 to 23 of the pre-neurotensin represent the so-called. Signal sequence.
• proneurotensin occurs predominantly in the central nervous system (CNS) and in special enteroendocrine cells, the so-called N-cells in the distal small intestine as well as in nerve fibers, which pass through the intestinal tract (13).
• CNS central nervous system
• N-cells in the distal small intestine as well as in nerve fibers, which pass through the intestinal tract (13).
• PNT and NT were also detected in the adrenals and cardiac tissue (24, 23).
• NT neurotensin
• the biologically active neurotensin (NT) formed from the proneurotensin was detected in various mammalian tissues and discussed in connection with numerous different functions. Neurotensin plays an important role in the brain as a neurotransmitter and has analgesic and thermoregulatory effects (6; 8). In addition, NT regulates the release of the neurotransmitters acetylcholine and glutamate (19; 18), stimulates the secretion of pituitary hormones (29), and influences dopamine neurotransmission (18), suggesting a link to Parkinson's disease (Fig. 28).
• neurotensin stimulates the secretion of pancreatic hormones, such as insulin and glucagon, inhibits the secretion of gastric acid and stimulates the digestive motility (33).
• pancreatic hormones such as insulin and glucagon
• gastric acid stimulates the digestive motility (33).
• NT promotes the growth of gastrointestinal tissue such as pancreatic, colon and small intestinal tissue (12; 36; 10), suggesting that NT contributes to the development of tumors in these tissues.
• Neuromedin N (SEQ ID NO: 3), formed from the same precursor peptide is less well characterized. Presumably, it has similar effects to neurotensin, as both have similarities in their amino acid sequence and bind to the same receptors (33).
• radioimmunoassay-type assays were used without exception, in which the competition of the neurotensin to be determined in the sample with an added labeled neurotensin or neurotensin fragment is determined around the binding sites of a single antibody which has specific binding with neurotensin or neurotensin .
• Measurements of NMN levels were made in a similar manner.
• Neurotensin determinations in blood samples have hitherto not been included in routine medical diagnostics.
• a first major reason for this is a lack of stability of neurotensin concentrations in blood samples ex vivo at ambient temperature (20) (half-life of only 3-4 h) for routine measurements, in combination with existing laboratory logis- tics (from sampling to plasma collection, transport to the laboratory) Laboratory until the implementation of appropriate laboratory tests) so far the use of Neurotensin in routine diagnostics prevented.
• a second reason lies in the known short half-life of only 2 to 6 minutes for neurotensin in blood in vivo, which is attributed to rapid and effective binding to neurotensin receptors and / or degradation of the neurotensin (3) and a potential one burdened with difficult to estimate time dependence.
• NT levels in blood samples do not provide a measure of the overall stimulus on a stimulating, z. B. feeding, biosynthesis and release of NT into the circulation by the time of measurement, but rather represent a random transit state in which NT elimination by receptor binding and degradation greatly dilutes biosynthesis and release information into the circulation.
• an immunodiagnostic assay for determining the release of neurotensin into the mammalian circulation which in its most general form according to claim 1 is characterized in that in a serum or plasma sample of a mammalian subject, selective immunoreactivity of the N terminal portion of a mammalian proneurotensin (PNT immunoreactivity) other than neurotensin or neuromedin N immunoreactivity.
• PNT immunoreactivity selective immunoreactivity of the N terminal portion of a mammalian proneurotensin
• immunoreactivity of the N-terminal part of a mammalian proneurotensin means an immunoreactivity ranging from the amino acid in position 24 (position 1 after the signal peptide) to the third last amino acid before the first amino acid of the neuromedin N (Lys) of the j Mammalian Preproneurotensin is localized.
• the range mentioned includes at least the respective amino acids 24 to 139 or 140, d. H. a sequence as shown for the human peptide in SEQ ID NO: 6.
• FIG. 1 Results of the measurement of the ex vivo stability of the immunoreactivity, which can be assigned to human PNT N-terminal sequences, in serum, EDTA plasma and heparin plasma samples obtained from test subject blood for periods of up to 7 days;
• Figure 2 The standard curve of a PNT sandwich assay, as described in more detail in the experimental section;
• Figure 3 The results of the measurement of PNT immunoreactivity in plasma in normal nutritionally normal persons and artificially nourished patients by means of described sandwich assays;
• FIG. 4 shows the results of the measurement of temporal changes in the PNT immunoreactivity in the plasma of normally fed normal persons as a function of the food intake by means of the described sandwich assay
• Figure 5 The results of the measurement of PNT immunoreactivity in plasma in normal dietary subjects, patients with inflammatory bowel disease (Crohn's disease / ulcerative colitis) and hepatitis patients using the sandwich assay described.
• Each peptide was additionally provided with an amino-terminal cysteine residue (CysO).
• peptides PNT3 (SEQ ID NO: 4) and PNT4 (SEQ ID NO: 5) supplemented by N-terminal cysteine residues were conjugated to Limulus polyphemus hemocyanin and used to immunize rabbits by standard methods, of which Antisera to the PNT-peptide conjugates were recovered.
• the polyclonal antibodies of the rabbit antisera were purified by ligand-specific affinity purification.
• the Cys (0) peptides PNT3 (SEQ ID NO: 4) and PNT4 (SEQ ID NO: 5) were first coupled to SuIfoLink gel Pierce (Boston, USA). The binding was carried out according to the instructions of the manufacturer as follows:
• Polycarbonate columns (15mm x 80mm) were filled with 5ml affinity matrix. After equilibration of the columns with PBS (136 mM NaCl, 1.5 mM KH 2 PO 4 , 20, 4 mM Na 2 HPO 4 * 2H 2 O, 2.7 mM KCl, pH 7, 2), 5 mg of the respective o. g. Peptides were weighed, dissolved in PBS, added to the sealed columns, and the gel material was homogenized by panning. After 15 minutes incubation at room temperature and settling of the gel material, the columns were washed with 5 times 3 ml PBS.
• PBS 136 mM NaCl, 1.5 mM KH 2 PO 4 , 20, 4 mM Na 2 HPO 4 * 2H 2 O, 2.7 mM KCl, pH 7, 2
• 5 mg of the respective o. g. Peptides were weighed, dissolved in PBS, added to the sealed columns, and the gel material was homogenized by panning. After 15
• the gel material was mixed with 25 ml of the respective rabbit Mix antiserum pools and incubate overnight at room temperature with gentle swirling.
• the serum-gel mixtures were transferred to polycarbonate columns and excess serum was removed.
• the columns were then washed with 250 ml of PBS to remove unbound serum proteins.
• the desorption of the bound antibodies was carried out by elution of the column with 5OmM citric acid (pH 2, 2).
• the eluate was collected in fractions aImI.
• the protein concentration of each fraction was determined using the BCA protein assay kit from Perbio (Bonn, Germany) and fractions with a protein content> 1 mg / ml were combined.
• the affinity-purified antibodies were rebuffered by dialysis in PBS, the protein content was determined again. It was then stored at 4 ° C.
• the affinity-purified polyclonal rabbit antibody to the peptide PNT3 was immobilized on polystyrene tubes (Startups, 12 mm ⁇ 75 mm, Greiner, Germany). For this, the antibody solution was diluted to a protein concentration of 6.7 ⁇ g / ml with PBS, and of these, 300 ⁇ l per tube were pipetted (corresponds to 2 ⁇ g of antibody per tube). These were incubated for 20 h at room temperature and then washed 3 times with 4 ml of PBS. Until further use, the tubes were stored at 4 ° C.
• the affinity-purified polyclonal rabbit antibody to PNT4 (1 mg / ml in PBS) was luminescently labeled with acridinium ester N-hydroxy-succinimide (1 mg / ml in acetonitrile, InVent, Hennigsdorf, Germany).
• acridinium ester N-hydroxy-succinimide (1 mg / ml in acetonitrile, InVent, Hennigsdorf, Germany).
• 200 ⁇ l of antibody were mixed with 4 ⁇ l of acridinium ester, incubated for 20 minutes, and free acridinium ester bins were added. fertilize were saturated by addition of 40 ⁇ l of 5OmM glycine solution.
• the labeling batch was separated from free acridinium ester by HPLC on a BioSil 400 gel filtration column (BioRad, Kunststoff, Germany). The eluent used was PBS.
• each antibody-coated tube 50 ⁇ l of a plasma sample to be examined and 150 ⁇ l of assay buffer (PBS buffer, 10 mM EDTA) were pipetted and incubated for 16 h at room temperature. Subsequently, the tubes were washed 5 times with j e ImI PBS. To the tubes was then added 20 ng of the labeled antibody (2.1.) (In lOO ⁇ l PBS buffer, 10 mM EDTA). The tubes were incubated for 2 h at RT and then unbound tracer antibody was removed by washing 5 times with ImI PBS each time.
• PBS buffer 10 mM EDTA
• Labeled antibody bound to the tube was quantified by luminescence measurement in a commercial luminometer (Berthold LB 952T / 16).
• EDTA plasmas from apparently healthy humans were screened for PNT, and plasmas with the highest PNT immunoreactivities were pooled. Several dilution steps with horse serum (Sigma, Germany) were prepared from this plasma pool. The highest standard (undiluted EDTA plasma) was assigned a notional concentration of 100 units / ml.
• FIG. 2 shows a PNT standard curve. The analytical sensitivity of the proneurotensin assay is approx. 1 U / ml. P2006 / 000659
• the measurement of PNT immunoreactivity can thus be used instead of the mature neurotensin for the assessment of the metabolic status of a patient / a subject.
• an amount of nutrients defined individual stimulatability can sober of PNT-release as the difference ⁇ of a measurement value at a predetermined time after the food intake and a base value (e .g. PNT 3h to near run GSA u f n ah m e and PNT ) or as a ratio or factor of such values.
• Table 2 shows an example of a corresponding evaluation.
• PNT B PNT immunoreactivity after 18 h fast (base value)
• PNT 5rain PNT immunoreactivity 5 min after food intake
• PNT 3h PNT immunoreactivity 3 h after ingestion 4.3. Determination of PNT concentrations in the circulation of artificially nourished patients
• a serum mixed sample of 10 individual sera of 100 ml with a relative concentration of 32 U / ml was mixed with Na-EDTA (final concentration 10 mM) and then filtered through an O, 2 ⁇ m filter.
• the treated serum sample was mixed six times in succession pumped at 4 0 C and a flow rate of 2 ml / min over the affinity column. Subsequently, the column was washed with 50 ml PBS and the bound PNT immunoreactivity with a 5OmM glycine / HCl solution (pH 2, 0) eluted.
• the column effluent was monitored continuously for absorbance at 280nm and the protein fraction eluted from the glycine / HCl solution (final volume 3ml) was further analyzed by HPLC.
• Fraction 67 which had positive PNT immunoreactivity, was dried by gassing with nitrogen. Subsequently, the samples were analyzed by mass spectrometry.
• the peptide was purified by protease Glu-C or. Trypsin digested, the resulting fragments were obtained in known manner via SMART-HPLC and then analyzed by mass spectrometry.
• Amino acids 24-140 of known preproneurotensin 1-170 (SEQ ID NO: 1). This has been shown to circulate a proneurotensin peptide in the blood of humans that comprises 117 amino acids and can be described as preproneurotensin 24-140. There was no suggestion that the species immunoreacted by the method described still contained the C-terminal mature peptides neuromedin N and neurotensin.
• Cigarette smoking potentiates fat-induced elevation of neurotensin-like immunoreactivity in human plasma. Acta Physiologica Scandinavia 121: 181-184

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Endocrinology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=36263813

Family Applications (1)

Country Status (6)

Cited By (2)

Families Citing this family (34)

Family Cites Families (14)

• 2005
  • 2005-01-26 DE DE102005003687A patent/DE102005003687A1/de not_active Withdrawn
• 2006
  • 2006-01-25 AT AT06706415T patent/ATE433116T1/de not_active IP Right Cessation
  • 2006-01-25 US US11/814,850 patent/US8637256B2/en active Active
  • 2006-01-25 JP JP2007552570A patent/JP5133066B2/ja active Active
  • 2006-01-25 EP EP06706415A patent/EP1769250B1/de active Active
  • 2006-01-25 DE DE502006003868T patent/DE502006003868D1/de active Active
  • 2006-01-25 WO PCT/EP2006/000659 patent/WO2006079528A1/de active Application Filing
• 2012
  • 2012-02-22 US US13/402,551 patent/US20120231475A1/en not_active Abandoned

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events